Enzymatic substrates for detecting Pseudomonas aeruginas

ABSTRACT

An enzyme substrate which includes a target portion and a marker portion, hydrolysis of the substrate leading to separation of the target portion from the marker portion, and the target portion being specific for the enzyme activity being assayed. A formulation containing at least one such substrate, and a method for detecting the bacterial species  Pseudomonas aeruginosa  is also disclosed. The target portion of the substrate is specific for β-alanine aminopeptidase activity and the marker portion is a compound which reveals whether a hydrolysis reaction has taken place or not. The invention is particularly applicable in the field of diagnosis.

This application is a U.S. National Stage of International applicationPCT/FR00/03398, filed Dec. 6, 2000, and published on Jun. 14, 2001 inthe French Language which claims priority to French Application 99/15312filed Dec. 6, 1999.

This invention concerns a substrate which can be used in a test based ondetection of the activity of the enzyme β-alanine aminopeptidase. Italso concerns a formulation containing at least one such substrate, anda method for detecting the bacterial species Pseudomonas aeruginosa.

For many years, special substrates have been used to determine whetherenzymatic activities typical of microorganisms are present or not.Through the use of specific substrates, it is possible-on the basis ofwhether a reaction takes place or not-to characterize the nature of agenus of microorganisms, or distinguish between different strains and/orspecies belonging to a given genus.

Synthetic enzyme substrates are made up of two different parts: thefirst part is specific to the enzyme activity being tested for and willhereafter be referred to as the target part; the second part acts as amarker and will hereafter be referred to as the marker part.

Such special substrates may be either fluorescent or chromogenic. Infact, the second marker part or the product of its reaction with one ormore other compounds becomes fluorescent or chromogenic when it is nolonger associated with the first target part (in this context, refer toPatent Application PCT/FR99/00781 filed on behalf of the applicant).

Prior art does not provide any simple test to characterize the speciesPseudomonas aeruginosa, although this species—by virtue of its potentpathogenicity, its widespread high incidence, and its role in manynosocomial infections—is still among the most commonly sought for.

In accordance with this invention, a range of substrates based onβ-Alanine is proposed, all of which will facilitate reliable detectionof Pseudomonas aeruginosa. The invention also concerns a formulationcontaining at least one such substrate, and a method for detectingPseudomonas aeruginosa.

Certainly, β-Alanine is a we characterized substrate. Thus, in a paperby Kasafirek, Evzen et al., entitled “Role of amino acid residues inchromogenic substrates cleaved by pancreatic elastase”, Collect. Czech.Chem. Commun. (1987), 52(6), 1625-33, mention is made of chromogenicsubstrates such as β-Alanine p-Nitroaniline (synthetic pathway given onpage 1631).

In an article published by J. B. Suszkiw, A. S. Brecher, on “BrainAminoacyl Arylamidase. Further Purification of the Soluble Bovine Enzymeand Studies on Substrate Specificity and Possible Active-Site Residues”,Biochemistry, vol. 9, no. 20 (1970), pages 4008-4017, a method isproposed for the purification from bovine brain tissue of a solubleArylamidase, β-Alanine β-naphthylamine (synthetic pathway given inparagraph 2 on age 4010).

In this case too, the target is completely different from that which weare proposing.

Patent application WO-A-96/40980 describes a method and a formulationfor detecting the existence of and measuring the overall density ofviable bacteria in foodstuffs. This involves using various substances,including N-Acetyl-L-phenylalanyl-L-arginine-7-amido-4-methylcoumarinhydrochloride.

However, the main objective in WO-A-96/40980 is not specificity for asingle species (e.g. Pseudomonas aeruginosa) but rather to assay allbacteria present. In order to do so many different substrates are used,none of which are capable of detecting Pseudomonas aeruginosa-specificβ-alanine aminopeptidase activity because the list given includes manydifferent species and, with respect to the genus Pseudomonas, only twospecies are mentioned, namely Pseudomonas fluorescens and Pseudomonasputida, the former being easily distinguished because Pseudomonasaeruginosa naturally gives green colonies due to its production ofpyoverdine, and the latter species does not express any βalanineaminopeptidase activity.

To this effect, this invention concerns an enzyme substrate made up ofone target part and one marker part, hydrolysis of this substrateleading to separation of the marker part and the target part, saidtarget part being specific for the enzyme activity being assayed,characterized in that this target part is acted upon by the enzymeβ-alanine aminopeptidase derived from at least one Pseudomonasaeruginosa microorganism, and the marker part is a molecule whichreveals whether a hydrolysis reaction has taken place or not.

According to a preferred embodiment, the target part consists ofβ-Alanine or a derivative thereof, and the marker part is a fluorescentor chromogenic molecule.

Still according to a preferred embodiment, the substrate has thefollowing formula:H₂N—CH₂—CH₂—CO—NH—R,in which H₂N—CH₂—CH₂—CO— is the target part and —NH—R is the chromogenicor fluorescent marker part of the substrate.

Again according to a preferred embodiment, the marker part consists of:

-   β-Naphthylamine,-   An aminomethylcoumarin, such as 7-amino-4-methyl-coumarin,-   an Aminobenzene derivative, such as 4-amino-2,6-dichlorophenol,-   p-Nitroaniline, or-   2-amino-indoxyl or 3-amino-indoxyl.

The invention also concerns a formulation for detecting at least onestrain and/or species of microorganism which includes at least onesubstrate as described above plus a culture medium.

According to a modification, a formulation for detecting at least onestrain and one species of microorganism, or at least two strains or twospecies of microorganism, which contains at least one substrate asdescribed above, plus at least one other substrate and a culture medium.

According to a modification, the culture medium contains a developer.

In this case, the developer in the culture medium is:

-   a diazonium salt, when the marker released is β-Naphthylamine, or-   3,5-dihydroxy-2-naphthoic acid, when the marker part released is an    Aminobenzene derivative.

Preferably, the formulation is in the form of a liquid broth or asemi-solid agar medium.

Finally, the invention concerns a method for detecting the bacterialspecies Pseudomonas aeruginosa, the method consisting in:

-   bringing at least one substrate capable of detecting β-alanine    aminopeptidase enzyme activity into contact with a sample suspected    of containing at least one Pseudomonas aeruginosa microorganism, and-   monitoring for the appearance of colored and/or fluorescent    reactions.

According to a modification, the detection method for the bacterialspecies Pseudomonas aeruginosa consists in:

-   bringing at least one substrate capable of detecting β-alanine    aminopeptidase enzyme activity into contact with a sample suspected    of containing at least one Pseudomonas aeruginosa microorganism,-   bringing at least one substrate capable of detecting the activity of    some enzyme other than β-alanine aminopeptidase into contact with a    sample suspected of containing at least one microorganism other than    Pseudomonas aeruginosa, this other enzyme activity making it    possible to distinguish Pseudomonas aeruginosa from this other    microorganism, and-   monitoring for the appearance of colored and/or fluorescent    reactions.

In both examples above, the culture medium is semi-solid.

In this case, some product is added to the culture medium to inhibit thediffusion of the color(s) obtained.

This invention concerns the exploitation of a biological parameter whichis specific to most strains of the bacterial species Pseudomonasaeruginosa, namely β-alanine aminopeptidase activity. This activity isonly found in a very small number of other species.

The invention consists in combining a suitable marker part with a targetpart which is specific for this enzyme, namely β-Alanine. Suitablemarker parts to reveal this activity are already in existence but theyhave not hitherto been used in the detection of Pseudomonas aeruginosabecause the link between this species and this enzyme activity has neverpreviously been described.

Thus, the first family of substrates consists of molecules with aNaphthylamine as the marker part. One of the formulae—corresponding tothe substrate β-alanyl-β-naphthylamide—is:

in which R₂, R₃, R₄, R₅, R₆, R₇, and R₈ represent an atom of hydrogen,bromine, chlorine or iodine, or a group such as —OH, —CH₃, —CH₂CH₃,—OCH₃, —OCH₂CH₃ or COOH at each of these positions. β-Naphtylamine canbe used to detect β-alanine aminopeptidase activity, as described in theDoctoral/Engineering Dissertation defended by Mr. Daniel Monget at theUniversité Claude Bernard—Lyon I on Jul. 4, 1978 (Order n° 297). Sincethis type of molecule is colorless, members of this family have to beused in conjunction with some kind of developer, such as a diazoniumsalt.

The second family of substrates in the background art is constituted bycompounds in which the marker part is based on aminomethylcoumarin whichbecomes fluorescent on release from the target part. The general formulafor this family of substrates can be deduced from the following specificformula which is that of β-Alanyl-7-amido-4- methylcoumarin:

in which R₉, R₁₂, R₁₃, R₁₄ and R₁₅ represent an atom of hydrogen,bromine, chlorine or iodine, or a group such as —OH, —CH₃, —CH₂CH₃,—OCH₃, —OCH₂CH₃ or COOH at each of these positions. This type ofsubstrate does not lend itself to use in semi-solid media and is morecommonly used in liquid broth.

The third family of substrates is constituted byβ-Alanyl-4-amino-2,6-dichlorophenol in which the marker part is2,6-Dichloroaminophenol which has the following formula:

in which R₁₆ and R₁₇ represent an atom of hydrogen, bromine, chlorine oriodine, or a group such as —OH, —CH₃, —CH₂CH₃, —OCH₃, —OCH₂CH₃ or COOHat each of these positions. This type of compound has already beencovered in patent application WO-A-99/51767, filed by the applicant, andrequires the presence of a dry developer such as 3,5Dihydroxy-2-naphthoic acid. This same patent application (WO-A-99/51767)also shows that it is possible to use other compounds in which the twochlorine atoms and the hydroxyl group are substituted by an atom ofhydrogen, bromine, chlorine or iodine, or a group such as —OH, —CH₃,—CH₂CH₃, —OCH₃, —OCH₂CH₃ or COOH at each of these positions.

The fourth family of substrates is based on β-Alanyl p-nitroanilide andits derivatives in which p-Nitroaniline acts as the marker part. Thegeneral formula for this type of substrate is:

in which R₁₈, R₁₉, R₂₀ and R₂₁ represent an atom of hydrogen, bromine,chlorine or iodine, or a group such as —OH, —CH₃, —CH₂CH₃, —OCH₃,—OCH₂CH₃ or COOH at each of these positions. This product does notrequire the presence of any developer.

In the fifth family of substrates, 2-Amino-indole or 3-Amino-indole actsas the marker part. These substrates have the following formulae: for2-Amino-indole:

and for 3-Amino-indole:

All these substrates are chromogenic apart from the second family whichis fluorescent, and all correspond to compounds which can be used toidentify the activity of the β-alanine aminopeptidase of Pseudomonasaeruginosa. This parameter can be used to clearly distinguish thispathogen from other, closely related species of Gram negative bacilli ina clinical setting.

1°) Substrates Used:

The enzyme substrates used had the following formula:H₂N—CH₂—CH₂—CO—NH—R,in which H₂N—CH₂—CH₂—COOH is β-Alanine, i.e. the target part of thesubstrate; and —NH₂—R is chromogenic, i.e. the marker part of saidsubstrate.

Four substrates were tested, corresponding to the first four families ofsubstrates mentioned above. They were:

-   β-Alanyl-β-naphtylamide, which is commercially available as product    reference K1035 from the Bachem company (Bubendorf, Switzerland),-   β-Alanyl-7-amido-4-methylcoumarin, which is commercially available    as product reference I1030 from the Bachem company (Bubendorf,    Switzerland),-   β-Alanyl-4-amino-2,6-dichlorophenol, the synthetic pathway for which    is given below, and-   β-Alanine p-nitroanilide, the synthetic pathway for which is given    below.

β-Alanyl-4-amino-2,6-dichlorophenol was synthesized fromN-Tert-butoxycarboxyl-β-alanine (1.89 g, 10 mmol). TheN-Tert-butoxycarboxyl-β-alanine was dissolved in anhydrousTetrahydrofurane (HPLC grade, 30 ml). The resultant solution was cooledto 0° C. in a ice-salt bath, and then N-methylmorpholine (2.02 g, 20mmol) was added. The temperature was lowered to below −10° C. beforeslowly adding Isobutyl chloroformate (2.74 g, 20 mmol) without lettingthe temperature rise above −8° C. To the resultant anhydrous suspensionwas then added a cold solution of 4-Amino-2,6-dichlorophenol (1.78 g, 10mmol) dissolved in Dimethylformamide and treated with N-methylmorpholine(4.08 g, 40 mmol) to release the free base. This solution was kept belowroom temperature before the above-mentioned addition operation. Theresultant reaction mixture was stirred in an ice-salt bath for one hour,and thereafter at room temperature for a further five hours. Thin layerchromatography showed that conversion to the amide was almost 100%. Thesuspension was filtered to remove the N-Methylmorpholine hydrochlorideand the residue was washed by adding Tetrahydrofurane. Next, thetetrahydrofurane and some of the dimethylformamide were removed using arotary evaporator and the resultant solution was poured into icy waterwith vigorous shaking. The gray precipitate was recovered, thoroughlywashed and dried in a vacuum oven at 30-40° C. After recrystallizationfrom methanol with a little added water, the yield was 1.72 g of small,white crystals. This product wasN-β-t-BOC-β-alanyl-4-amino-2,6-dichlorophenol which was subsequentlydissolved in the smallest possible volume of hydrochloric acid-saturatedEthyl acetate. After two hours at about 16° C., the viscous solution wasgently poured into 250 ml of anhydrous Diethyl ether. After four hours,the powdery solid was recovered by vacuum filtration and then washed inDiethyl ether. Finally, the product was dried in a vacuum drier andstored in a tightly closed container in order to prevent it absorbingmoisture.

β-Alanine p-nitroanilide was synthesized in the following way. A mass of2.76 grammes (g), i.e. 20 millimoles (mmol) of 4-Nitroaniline wasdissolved by shaking in 40 milliliters (ml) of dry pyridine which hadbeen cooled to below 12° C. In parallel, Phosphorus trichloride (1 mlredistilled) was added to 12 ml of pyridine (also at a temperatuere ofbelow 12° C.). Both solutions were then cooled to below 16° C., and thenthe Phosphorus trichloride-containing solution was added with shaking tothe Nitroaniline-containing solution. Mixing was carried out at atemperature of between below 14 and below 16° C. for at least 30 minutes(min) and then for the same amount of time at room temperature. Next,dry Benzyloxycarbonyl-β-Alanine (20 mmol, i.e. 4.46 g) was addedstepwise over at least 5 min to the resultant solution, stirringthroughout. Stirring was maintained for 14 hours (h) at between 30 and40° C. Subsequently, the temperature was raised to 50° C. and stirringcontinued for a further 8 h. Thin layer chromatography of a dilutedsample showed that most of the starting materials had been convertedinto the β-aminoacyl derivative. The pyridine was removed by rotaryevaporation at 50° C. and the residual viscous oil was shaken vigorouslywith crushed ice and ethanol. This yielded a yellowish solid, some ofwhich was deposited on the walls of the vessel, the rest making up, acrystalline mass. The solid was recovered, filtered with aspiration andwashed with water. The product was air-dried and recrystallized from hotethanol. The dry product weighed 4.3 g and was shown to be pure by thinlayer chromatography (TLC) using silica gel plates (with ethylacetate-toluene as the solvent). The yield was 62.7%. The protectionconstituted by the Benzyloxycarbonyl, present in theBenzyloxycarbonyl-β-alanine-p-nitroanilide, was then removed. To dothis, 2.15 g of this material were dissolved in the smallest possiblevolume of hot glacial acetic acid with stirring. After this mixture hadbeen cooled down to 25° C., an equal volume of 30% hydrobromic acid inglacial acetic acid was added with stirring. After 1 h 30 min, theresultant viscous solution was slowly poured into 400 ml of anhydrousDiethyl ether, with vigorous stirring throughout. The resultant whitesuspension was filtered and the residue washed with several aliquots ofanhydrous ether before drying overnight in a vacuum drier. The yield was1.50 g of β-Alanine-p-nitroanilide in the form of the bromide salt.

2°) Detection of β-Alanine Aminopeptidase Enzyme Activity Using VariousEnzyme Substrates

A—Testing β-Alanyl-β-naphthylamide and the Selectivity of its TargetPart:

A number of species (a total of five strains per species unlessotherwise specified) were tested in the presence ofβ-alanyl-β-naphthylamide using APIZYM-type test strips from bioMérieux(La Balme, France). Experiments were carried out with bacterialsuspensions at a density of 4 McFarland units (McF) in phosphate buffer(0.01 M, pH 7.5). On the McFarland Scale which measures bacterialdensity, 4 McF corresponds to 12.10⁸ bacteria per milliliter. For everystrain tested, 80 microliters of the relevant suspension were added toeach well on a strip. The strips were incubated at 37° C. for 4 h. Thepresence of β-alanine aminopeptidase was detected by adding a drop ofZYMB (Fast Blue BB, a diazonium salt) which was obtained from bioMérieux(Product Reference 70480). If the reaction was positive, the solutionturned orange. The results were as follows. The species in the followinglist all gave a negative result, i.e. no β-alanine aminopeptidaseactivity was detected in the following species:

-   Escherichia coli,-   Shigella spp. (4 strains),-   Shigella sonnei,-   Edwarsiella tarda (2 strains),-   Salmonella choleraesuis,-   Salmonella typhi,-   Salmonella spp.,-   Salmonella paratyphi A,-   Salmonella gallinarum (3 strains),-   Salmonella arizonae,-   Citrobacter freundii,-   Citrobacter diversus,-   Klebsiella pneumoniae, spp. pneumoniae,-   Klebsiella pneumoniae, spp. oxytoca,-   Klebsiella ozaenae,-   Klebsiella rhinoscleromatis (3 strains),-   Hafnia alvei,-   Enterobacter aerogenes,-   Enterobacter cloacae,-   Enterobacter sakazakii,-   Enterobacter gergoviae,-   Serratia marcescens,-   Serratia odorifera,-   Serratia fonticola,-   Serratia plymuthica,-   Proteus vulgaris,-   Proteus mirabilis,-   Proteus morganii,-   Proteus rettgeri,-   Providencia alcalifaciens,-   Providencia stuartii,-   Yersinia enterocolitica,-   Yersinia pseudotuberculosis,-   Pseudomonas putida,-   Comamonas acidovorans,-   Comamonas testosteroni,-   Pseudomonas alcaligenes,-   Pseudomonas stutzeri,-   Shewanella putrefaciens,-   Stenotrophomonas maltophilia,-   Brevundimonas diminuta,-   Brevundimonas vesicularis,-   Ralstonia picketti,-   Pseudomonas luteola,-   Pseudomonas oryzihabitans,-   Sphingomonas paucimobilis,-   Sphingobacterium multivorum,-   Alcaligenes faecalis/odorans,-   Achroniobacter xylosoxidans spp. denitrificans,-   Achromobacter xylosoxidans, spp. xylosoxidans,-   Bordetella bronchiseptica,-   Oligella ureolytica (2 strains),-   CDC IV C₂,-   Aeromonas hydrophila,-   Plesiomonas shigelloïdes,-   Vibrio alginolyticus,-   Vibrio parahaemolyticus (4 strains),-   Chryseobacterium meningosepticum,-   Empedobacter brevis (3 strains),-   Chryseobacterium balustinum (1 strain),-   Chryseobacterium indologenes,-   Weeksella virosa (4 strains),-   Myroïdes spp.,-   Pasteurella gallinarum (1 strain),-   Pasteurella multocida,-   Pasteurella pneumotropica (4 strains),-   Actinobacillus ureae (1 strain),-   Pasteurella haemolytica,-   Pasteurella aerogenes,-   proches de Pasteurella (4 strains),-   Psychrobacter phenylpyruvicus (4 strains),-   Moraxella paraphenylpyruvica (3 strains),-   Moraxella lacunata (4 strains),-   Moraxella lac. sp. liquefaciens (7 strains),-   Moraxella non liquefaciens (13 strains),-   Moraxella bovis (4 strains),-   Moraxella osloensis (18 strains), and-   Actinobacillus spp.

In contrast, the species in the following list all gave a positiveresult, i.e. β-alanine aminopeptidase activity was detected in thefollowing species:

-   Serratia liquefaciens,-   Pseudomonas aeruginosa,-   Pseudomonas fluorescens,-   Burkholderia cepacia,-   Pseudomonas mendocina., and-   Ochrobactrum anthropi.

CDC is an acronym for the Center for Disease Control. CDCclassifications concern groups of strains which have not yet beenassigned a species name.

Therefore, the substrate β-alanyl -β-naphthylamide and specifically itstarget part β-alanine-is highly selective in that only six (6) speciesout of the seventy nine (79) tested (i.e. a fraction of only 7.1%) werefound to posses β-alanine aminopeptidase activity.

On semi-solid medium, Pseudomonas aeruginosa can easily be distinguishedfrom the other five species which give a positive result by virtue ofthe green tint of its colonies (which is due to the production ofpyoverdine).

B—Testing Substrate β-Alanyl-7-amido-4-methylcoumarin and Assessing itsEfficacy with Respect to the Detection of Strains of Pseudomonasaeruginosa:

The substrate β-Alanyl-7-amido-4-methylcoumarin was tested in thefollowing way. Strains were isolated using the following media frombioMérieux (France):

-   Columbia Sheep's Blood,-   Trypticase Soy with Sheep's Blood, and-   MacConkey (used to isolate enteric bacilli).

The experiments were carried out using Vitek 2 cards (RegisteredTrademark, bioMérieux, Saint Louis, Mo., United States of America) withbacterial suspensions adjusted to a density of between 0.375 and 0.750McF.

Table 1 below shows the usefulness of the substrate tested for some ofthe species dealt with in the preceding section, as well as for someother species (e.g. Ochrobactrum anithropi).

Simple tests such as the Arginine Hydrolysis or Acetamide Tests can beused to distinguish between Pseudomonas aeruginosa and Ochrobactrumanthropi.

TABLE 1 Efficacy of the substrate β-Alanyl-7-amido-4-methylcoumarinNumber of Number of Number of strains negative positive BorderlineSPECIES tested results results results Shigella boydii 10 10 0 0Shigella dysenteriae 10 10 0 0 Shigella flexneri 10 10 0 0Chromobacterium 11  9 1 1 violaceum Chryseobacterium 10 10 0 0indologenes Ochrobactrum anthropi 11  0 11  0 Oligella urethralis 10 100 0 Pseudomonas aeruginosa 137   2 134  1 Ralstonia pickettii 10  8 2 0Pseudomonas mendocina 10  0 10  0 Pseudomonas stutzeri 10 10 0 0Comamonas testoteroni 10 10 0 0

Therefore, the substrate β-Alanyl-7-amido-4-methylcoumarin isparticularly effective when it comes to detecting the following species:

-   Ochrobactrum anthropi, since the percentage of positive results is    100%,-   Pseudomonas aeruginosa, since the percentage of positive results is    97.8%, and-   Pseudomonas mendocina, since the percentage of positive results is    100%,

The efficacy with respect to Pseudomonas aeruginosa is of particularinterest because a total of 137 strains were tested. The fact that threestrains gave either a negative or a borderline result is due tobiological variation within this species.

Some strains of other species were found to be able to hydrolyzeβ-Alanyl-7-amido-4-methylcoumarin, namely:

-   Ralstonia pickettii, since the percentage of positive results is    20%, and-   Chromobacterium violaceum, since the percentage of positive results    is 10%,    C—Testing substrate β-Alanyl-4-amino-2,6-dichlorophenol and    Assessing its Efficacy with Respect to the Detection of Strains of    Pseudomonas aeruginosa:

After isolation on Columbia Sheep's Blood agar, strains were tested onVitek 2 cards (Registered Trademark, bioMérieux, Saint Louis, Mo.,United States of America) in broth containing the substrateβ-Alanyl-4-amino-2,6-dichlorophenol together with3,5-dihydroxy-2-naphtoique and peptone, buffered to pH 8.5. Thebacterial suspensions used were at a density of 0.5 McF in saline (4.5 gNaCl/liter). Results were read visually after 22 to 24 hours ofincubation. The results of these experiments are presented in Table 2below.

TABLE 2 Efficacy of the substrate β-Alanyl-4-amino-2,6-dichlorophenolNumber of Number of Number strains negative positive SPECIES testedresults results Citrobacter amalonaticus 2 2 0 Citrobacter freundii 2 20 Citrobacter koseri 1 1 0 Enterobacter aerogenes 2 2 0 Enterobactercloacae 2 2 0 Enterobacter intermedius 1 1 0 Escherichia coli 2 2 0Proteus mirabilis 2 2 0 Proteus vulgaris 1 1 0 Providencia stuartii 1 10 Salmonella arizonae 1 1 0 Serratia liquefaciens 1 0 1 Serratiamarcescens 3 0 3 Klebsiella oxytoca 2 2 0 Klebsiella pneumoniae 3 3 0Achromobacter xylosoxydans 2 2 0 Acinetobacter baumannii 2 2 0 Aeromonashydrophila 2 2 0 Brevundimonas diminuta 2 2 0 Burkholderia vesicularis 11 0 Chryseobacterium 2 2 0 meningosepticum Ochrobactrum anthropi 1 0 1Moraxella non liquefaciens 2 2 0 Myroïdes spp. 1 1 0 Pseudomonasaeruginosa 13  0 13  Pseudomonas fluorescens 2 1 1 Pseudomonas mendocina1 0 1 Pseudomonas putida 1 1 0 Pseudomonas stutzeri 3 3 0 Ralstoniapickettii 1 1 0 Shewanella algae 1 1 0 Shewanella putrefaciens 1 1 0Sphingomonas paucimobilis 2 2 0 Stenotrophomonas maltophilia 2 2 0Streptococcus agalactiae 1 1 0 Streptococcus pyogenes 2 2 0 Enterococcusfaecalis 2 2 0 Enterococcus faecium 2 2 0 Vibrio vulnificus 1 1 0

With this substrate, β-Alanyl-4-amino-2,6-dichlorophenol, the resultsfor Pseudomonas aeruginosa, Serratia liquefaciens, Serratia marcescenswere still significant, and those for Pseudomonas mendocina andOchrobactrum anthropi fairly so. It may therefore be useful to combinethis substrate with another one which makes it possible to distinguishPseudomonas aeruginosa from other microorganisms which give a positivereaction (see paragraph 3). The pattern observed with Pseudomonasfluorescens is difficult to interpret and may be due to eithermethodological problems or genetic variability in this species.

Moreover, it should be noted that the presence of polyvinyl pyruvate(PVP) deepens the color in a positive reaction.

D—Testing Substrate β-Alanyl Para-nitroanilide:

Substantially the same methods were used as those described in theprevious Paragraph C except that the visual read-out was performed after19 hours. The substrate used was β-Alanyl p-nitroanilide. The resultsare summarized in Table 3 below.

TABLE 3 Efficacy of the substrate β-Alanyl-p-nitroanilide Number ofNumber of Number strains negative positive SPECIES tested resultsresults Citrobacter amalonaticus 2 2 0 Citrobacter freundii 2 2 0Citrobacter koseri 1 1 0 Enterobacter aerogenes 2 2 0 Enterobactercloacae 2 2 0 Enterobacter intermedius 1 1 0 Escherichia coli 2 2 0Proteus mirabilis 2 2 0 Proteus vulgaris 1 1 0 Providencia stuartii 1 10 Salmonella arizonae 1 1 0 Serratia liquefaciens 1 0 1 Serratiamarcescens 1 0 1 Klebsiella oxytoca 2 2 0 Klebsiella pneumoniae 2 2 0Achromobacter xylosoxydans 2 2 0 Acinetobacter baumannii 2 2 0 Aeromonashydrophila 2 2 0 Brevundimonas diminuta 1 1 0 Burkholderia vesicularis 11 0 Chryseobacterium 1 1 0 meningosepticum Moraxella non liquefaciens 22 0 Myroïdes spp. 1 1 0 Pseudomonas aeruginosa 4 0 4 Pseudomonas putida1 1 0 Pseudomonas stutzeri 3 3 0 Ralstonia pickettii 1 1 0 Shewanellaalgae 1 1 0 Sphingomonas paucimobilis 2 2 0 Stenotrophomonas maltophilia2 2 0 Vibrio vulnificus 1 1 0

These results are very revealing because Pseudomonas aeruginosa iseasily distinguished from other species which do not possess β-alanineaminopeptidase activity.

Therefore, substrates with a target part consisting of β-Alaninedesigned to detect β-alanine aminopeptidase activity are ideal fordetecting Pseudomonas aeruginosa, and a few other species (Serratialiquefaciens, Pseudomonas marcescens, Pseudomonas mendocina andOchrobactrum anthropi) which are far less commonly encountered in testsamples. In consequence, detection of this enzyme activity will mostoften be associated with the presence of Pseudomonas aeruginosa in thebiological material being tested. It is nevertheless possible—as will bedescribed in the next Paragraph—to distinguish Pseudomonas aeruginosafrom any of these other species which possess β-alanine aminopeptidaseactivity.

3°) Concomitant Use of Two Enzyme Substrates To Assay Two DifferentEnzyme Activities, One Of Which Is That Of β-Alanine Aminopeptidase:

Using two substrates to assay two different enzyme activities isparticularly useful. For example, the two different activities might bethose of β-glucosidase and β-alanine aminopeptidase.

As mentioned in Example A in Chapter 2, the two different speciesPseudomonas aeruginosa and Pseudomonas mendocina both express β-alanineaminopeptidase activity. These two species can however be distinguishedby assaying for β-glucosidase activity which is only expressed byPseudomonas mendocina. This activity can, for example, be assayed usingthe substrate 6-Chloro-3-indolyl-β-D-glycopyranoside which can beobtained from Biosynth (Staad, Switzerland).

These two species can be distinguished on semi-solid medium, asdescribed in the following.

Trypticase Soy agar was supplemented with the following:

-   200 mg of 6-Chloro-3-indolyl-β-D-glycopyranoside,-   50 mg of β-Alanyl-N,N′-dimethyl-p-phenylene-diamine, and-   15 mg of 3,5-Dihydroxy-2-naphthoic acid.

The medium was poured out and inoculated as follows:

-   10 dishes with pure cultures corresponding to five strains of    Pseudomonas aeruginosa and five of Pseudomonas mendocina, and-   2 dishes with a mixture of two strains, one being of Pseudomonas    aeruginosa and the other of Pseudomonas mendocina.

The Petri dishes were incubated at 35-37° C. After 18-24 hours ofincubation, the color of the colonies was recorded. Blue colonies with ablue halo correspond to Pseudomonas aeruginosa which only expressesalanine aminopeptidase activity; purple colonies with a blue halocorrespond to Pseudomonas mendocina which expresses both β-alanineaminopeptidase and β-glucosidase activities.

Thus, even if they are mixed together in the same specimen, these twodifferent species can be easily distinguished on the same semi-solidmedium. This example illustrates how it is possible to combine togetherin the same reaction medium different substrates specific for differentenzyme activities, including one according to this invention for thecharacterization of Pseudomonas aeruginosa.

1. An enzyme substrate comprising a target portion and a marker portion,said target portion and marker portion being separable by hydrolysis,said target portion being specific for the enzyme activity beingassayed, such that said target portion is acted upon by the enzymeβ-alanine aminopeptidase derived from at least one Pseudomonasaeruginosa microorganism, and the marker portion is a molecule whichreveals whether a hydrolysis reaction has taken place or not, whereinsaid substrate has the formula:H₂N—CH₂—CH₂—CO—NH—R, in which H₂N—CH₂—CH₂— CO is the target portion and—NH—R is the marker portion of the substrate.
 2. The substrate of claim1, wherein the marker portion is a fluorescent or chromogenic molecule.3. The substrate of claim 1, wherein said target portion is not actedupon by an enzyme of E. Coli.
 4. The substrate of claim 1, wherein themarker portion is selected from the group consisting of β-naphthylamine,an aminomethylcoumarin, an aminobenzene derivative, p-nitroaniline,2-amino-indoxyl, and 3-amnino-indoxyl.
 5. The substrate of claim 4,wherein said marker portion is 7-amino-4-methyl-coumarin.
 6. Thesubstrate of claim 4, wherein said marker portion is 4-amino-2,6-dichlorophenol.
 7. A formulation for detecting at least one strainand/or species of microorganism comprising at least one enzyme substratecomprising a target portion and a marker portion, said target portionand marker portion being separable by hydrolysis, said target portionbeing specific for the enzyme activity being assayed, such that saidtarget portion is acted upon by the enzyme β-alanine aminopeptidasederived from at least one Pseudomonas aeruginosa microorganism, and themarker portion is a molecule which reveals whether a hydrolysis reactionhas taken place or not, wherein said substrate has the formula:H₂N—CH₂—CH₂—CO—NH—R, in which H₂N—CH₂—CH₂—CO— is the target portion and—NH—R is the marker portion of the substrate, and a culture medium. 8.The formulation of claim 7, wherein said formulation is either in theform of a liquid broth or a semi-solid medium.
 9. The formulation ofclaim 7, wherein the culture medium comprises a developer which is adiazonium salt when the marker portion released is β-naphthylamine, orthe developer is 3, 5-dihydroxy-2-naphthoic acid when the marker portionreleased is an aminobenzene derivative.
 10. The formulation of claim 9,wherein said aminobenzene derivative is dichloro-amino-phenol.
 11. Aformulation for detecting at least one strain and one species ofmicroorganism, or at least two strains or two species of microorganism,comprising at least one enzyme substrate comprising a target portion anda marker portion, said target portion and marker portion being separableby hydrolysis, said target portion being specific for the enzymeactivity being assayed, such that said target portion is acted upon bythe enzyme β-alanine aminopeptidase derived from at least onePseudomonas aeruginosa microorganism, and the marker portion is amolecule which reveals whether a hydrolysis reaction has taken place ornot, wherein said substrate has the formula:H₂N—CH₂—CH₂—CO—NH—R, in which H₂N—CH₂—CH₂—CO— is the target portion and—NH—R is the marker portion of the substrate, a culture medium, and atleast one other substrate.
 12. A method for detection of Pseudomonasaeruginosa, comprising: contacting at least one substrate capable ofdetecting β-alanine aminopeptidase enzyme activity with a samplesuspected of containing at least one Pseudomonas aeruginosamicroorganism, and monitoring for the appearance of a colored and/orfluorescent signal from said sample and which correlates to saidβ-alanine aminopeptidase enzyme activity. wherein said substrate has theformula:H₂N—CH₂—CH₂—CO—NH—R,  in which H₂N—CH₂—CH₂—CO— is the target portion and—NH—R is the marker portion of the substrate.
 13. The method of claim12, wherein said sample comprises a culture medium which is semi-solid.14. The method of claim 13, wherein a compound is added to the culturemedium to inhibit diffusion of colored and/or fluorescent molecules. 15.A method for detection of Pseudomonas aeruginosa, comprising: contactingat least one substrate capable of detecting β-alanine aminopeptidaseenzyme activity with a sample suspected of containing at least onePseudomonas aeruginosa microorganism, contacting at least one substratecapable of detecting the activity of an enzyme other than β-alanineaminopeptidase with a sample suspected of containing at least onemicroorganism other than Pseudomonas aeruginosa, this other enzymeactivity making it possible to distinguish Pseudomonas aeruginosa fromsaid other microorganism, and monitoring for the appearance of a coloredand/or fluorescent signal from said sample and which correlates to saidβ-alanine peptidase enzyme activity. wherein said substrate has theformula:H₂N—CH₂—CH₂—CO—NH—R,  in which H₂N—CH₂—CH₂—CO— is the target Portion and—NH—R is the marker portion of the substrate.